Bone repair and regeneration represents an expanding, multi-billion dollar market addressed through the fields of oral/maxillofacial and orthopaedic surgery. Common cases involve bone loss due to trauma, tumor resection, revision surgery, developmental deformities and non-union fractures, and dental bone loss as a result of missing teeth and periodontal disease. Currently available treatment options (i.e., autografts, allografts, etc.) are far from ideal, often resulting in a limited degree of structural and functional recovery, as well as other serious complications. Bone tissue engineering (BTE) may serve as a superior alternative treatment. Successful BTE critically depends on an effective three-dimensional, biodegradable scaffold, and an adequate vascular supply. The overall objective of this study is to develop an optimized biodegradable scaffold, seeded with clinically relevant cells to promote enhanced bone regeneration and vascularization. This study focuses on poly(85 lactide-co-15 glycolide) (PLGA) microsphere scaffolds since they are biodegradable, osteocompatible, and mechanically compatible with human bone. Unfortunately, bone regeneration achieved with these microsphere scaffolds (pore size ~100 5m) is limited to the scaffold surfaces, due to failure to support sufficient mass transport of oxygen and nutrients, and neo-vascularization. Non-PLGA microsphere scaffolds with larger pore sizes (i.e., >400 5m), although not mechanically compatible with human bone regeneration, have been shown to ease these limitations, improve cell infiltration, and ultimately, allow for increased bone formation and vascularization throughout the entire scaffold. In addition, recent work has demonstrated that pre-vascularizing scaffolds in vitro by co-culturing two clinically relevant cell populations, peripheral blood derived - endothelial progenitor cells (EPCs) and bone marrow derived - mesenchymal stem cells (MSCs), enhances both bone formation and vascularization in vivo. We hypothesize that pre-vascularized, mechanically strong PLGA microsphere scaffolds with increased pore size (i.e., moderately-sized pores) will promote increased bone formation, by improving cell proliferation, mineralization and vascularization throughout the entire scaffold. We propose to achieve this main objective through a three- step process. First, we will design, fabricate and characterize (i.e., porosity, interconnectivity and mechanical strength) novel moderately-porous and mechanically strong PLGA microsphere scaffolds. Second, we will assess the ability of these moderately-porous PLGA microsphere scaffolds seeded with two clinically-relevant cell populations to demonstrate enhanced mineralization and the formation of primitive vascular networks compared to control scaffolds in vitro. Lastly, we will study the enhanced bone regeneration ability of our pre- vascularized moderately-porous PLGA microsphere scaffolds in vivo via a rabbit ulnar bone defect model. Our approach is designed to significantly advance the state-of-the-art in scaffold-based BTE through the development of a technique to enable fully functional and structural bone regeneration. PUBLIC HEALTH RELEVANCE: Bone replacement and repair in oral/maxillofacial and orthopaedic surgery represent a multi-billion dollar market, which is rapidly expanding due to increasing demand and improving treatment options. Scaffold-based bone regeneration, the most promising treatment option on the horizon, is substantially limited by insufficient vascularization. This study will develop a novel technique for enhanced scaffold-based bone regeneration and vascularization through the development of pre-vascularized, biodegradable and optimally-designed scaffolds.